the regulation of tgf signal transduction · signal transduction in metazoans and emphasize events...

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Transforming growth factor (TGF) pathways are implicated inmetazoan development, adult homeostasis and disease. TGFligands signal via receptor serine/threonine kinases thatphosphorylate, and activate, intracellular Smad effectors as wellas other signaling proteins. Oligomeric Smad complexesassociate with chromatin and regulate transcription, definingthe biological response of a cell to TGF family members.Signaling is modulated by negative-feedback regulation viainhibitory Smads. We review here the mechanisms of TGFsignal transduction in metazoans and emphasize events crucialfor embryonic development.

IntroductionThe human transforming growth factor (TGF) family consists of33 members, most of which encode dimeric, secreted polypeptidesthat control developmental processes, ranging from gastrulation andbody axis asymmetry to organ-specific morphogenesis and adulttissue homeostasis (reviewed by Derynck and Miyazono, 2008). Inaddition to TGFs, this family includes the bone morphogeneticproteins (BMPs), growth and differentiation factors (GDFs), activinsand nodal. The TGF family is conserved throughout metazoanevolution. At the cellular level, TGF family members regulate cellgrowth, differentiation, adhesion, migration and death, in adevelopmental context-dependent and cell type-specific manner. Forexample, TGF more often inhibits, but sometimes also stimulates,cell proliferation (reviewed by Yang and Moses, 2008). Furthermore,nodal signaling sometimes inhibits, whereas BMP promotes, celldifferentiation, as in stem cells (Watabe and Miyazono, 2009). AsTGF ligands act multifunctionally in numerous tissue types, theyalso play complex roles in various human diseases, ranging fromautoimmune to cardiovascular diseases and cancer (reviewed byGordon and Blobe, 2008; Massagué, 2008).

Here we review the core components of the TGF family and theirsignaling engines, as part of a Minifocus in this issue on TGFsignaling (see Box 1), and discuss emerging concepts concerning theregulatory mechanisms of TGF pathways at the receptor,cytoplasmic and nuclear level. We also highlight recent discoveriesthat are of particular developmental relevance.

The TGF familyThe development of the axes and the asymmetry of the animal bodydepends on the localized action of extracellular signals, such as theWnt, nodal and BMP ligands. Gradients of these ligands, theirextracellular regulators and the competence of receptors inresponding cells, play important roles during tissue morphogenesis(Affolter and Basler, 2007; Smith and Gurdon, 2004). TGF familymembers also contribute to tissue patterning and are importantregulators of stem cell self-renewal and differentiation (see Box 2)(De Robertis and Kuroda, 2004; Watabe and Miyazono, 2009).

The TGFmorphogens include numerous secreted and conservedpolypeptides (Table 1), which emerged at the onset of multicellular(metazoan) life (Huminiecki et al., 2009). Structurally, this familyis characterized by a specific three-dimensional fold and by aconserved number and spacing of cysteine residues in the C-terminus of the mature polypeptide (Derynck and Miyazono, 2008).The prototypic TGF isoforms (TGF1, 2, 3), and the relatedinhibin polypeptides that make up the activin and inhibinmembers, have nine characteristic cysteines, eight of which formfour intramolecular disulfide bridges, while one intermolecular bondlinks the two monomers. The inhibin polypeptides, BMPs andGDFs have seven cysteines, of which six form intramolecular andone intermolecular bridges. The lefty proteins, GDF3, GDF9 andBMP15A have six cysteines in their mature sequence and lack theintermolecular bridge between the two monomers. The lack ofcovalent dimers provides regulatory flexibility; for example, leftyforms non-covalent complexes with nodal and binds to theglycosylphosphatidylinositol (GPI)-anchored co-receptor of theepidermal growth factor-Cripto/FRL-1/Cryptic (EGF-CFC) family,leading to the inhibition of nodal signaling (Chen and Shen, 2004).

Xenopus laevis expresses TGFs, nodal, activins, BMPs andGDFs (De Robertis and Kuroda, 2004), and additional uniquefamily members, such as the mesoderm-inducer Derrière, and thesix nodal-related proteins XNR1-6 (Eimon and Harland, 2002;Onuma et al., 2002; Ramis et al., 2007). Drosophila melanogasterhas only seven TGF family members (Table 1). The BMP-likeligands Decapentaplegic (Dpp) and Screw (Scw) regulatedorsoventral pattering and the differentiation of imaginal discs, suchas the wing disc (Affolter and Basler, 2007; Serpe et al., 2005). TheBMP-like Glass bottom boat (Gbb) regulates brain and wing discdifferentiation (Bangi and Wharton, 2006; Goold and Davis, 2007).The activin-like dActivin (Act – FlyBase) and Dawdle (Daw)ligands have tissue-specific roles (for example, in the larval brain),whereas much remains to be understood about the functions ofMaverick, the GDF8 (myostatin)-like ligand, and of Myoglianin,which are expressed in endodermal and mesodermal cells (Lee-Hoeflich et al., 2005; Nguyen et al., 2000; Zhu et al., 2008). InCaenorhabditis elegans, the BMP-like DBL-1, and the TGF-likeDAF-7, regulate body length and the dauer pathway, a specialenvironmental adaptation of earthworms, respectively (Table 1)(reviewed by Savage-Dunn, 2005). The other three ligands, TIG-2,TIG-3 and UNC-129, are as yet unexplored.

Development 136, 3699-3714 (2009) doi:10.1242/dev.030338

The regulation of TGF signal transductionAristidis Moustakas and Carl-Henrik Heldin

Ludwig Institute for Cancer Research, Uppsala University, BMC, Box 595, SE-75124Uppsala, Sweden.

[email protected]; [email protected]

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Box 1. Minifocus on TGF signalingThis article is part of a Minifocus on TGF signaling. For further reading,please see the accompanying articles in this collection: ‘Theextracellular regulation of bone morphogenetic protein signaling’ byDavid Umulis, Michael O’Connor and Seth Blair (Umulis et al., 2009);‘Informatics approaches to understanding TGF pathway regulation’by Pascal Kahlem and Stuart Newfeld (Kahlem and Newfeld, 2009);and ‘TGF family signaling: novel insights in development and disease’,a review of a recent FASEB Summer Conference on TGF signaling byKristi Wharton and Rik Derynck (Wharton and Derynck, 2009).

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Although specification of body asymmetry is a fundamentalfunction of TGF-like proteins during early embryogenesis, theidentification of genes that encode a complete TGF pathway in theprimitive metazoan Trichoplax adhaerens, a two-cell-layered animalthat lacks obvious body asymmetry, suggests that these morphogensmight have played a fundamental role in the specification of themulticellularity that precedes body asymmetry during animalevolution (Huminiecki et al., 2009).

TGF secretion and extracellular regulationAll TGF ligands are synthesized as precursor proteins with a longerN-terminal pro-peptide followed by a shorter C-terminal maturepolypeptide (reviewed by ten Dijke and Arthur, 2007). Intermoleculardisulfide linkages pair dimers of these precursors via conservedcysteine residues in the pro-peptide and mature peptide sequence.While precursor proteins are in the secretory pathway, furin-likeproteases cleave the pro-peptide from the mature peptide. The TGFpro-peptide, called the latency-associated peptide (LAP), continues toscaffold the smaller mature peptide within its core, serving as achaperone during exocytosis of the complex. It also mediates thedeposition of TGF in the extracellular matrix (ECM) through itscovalent association with large secreted proteins called latent TGF-binding proteins (LTBPs), and with ECM proteins, such as fibronectinand fibrillin 1 (reviewed by Rifkin, 2005). Activation of the mature C-terminal dimeric ligands from their matrix-deposited, multi-protein‘cages’ relies on several proteases, including elastase (which cleavesfibrillin 1), BMP1/Tolloid family proteases (which cleave LTBPs),and matrix metalloproteases, such as MMP2 (which cleave TGFLAPs) (reviewed by ten Dijke and Arthur, 2007).

The ability of the TGF LAP to maintain the ligand in an inactivestate is conserved among some TGF family ligands, such as GDF8and GDF11 (Ge et al., 2005; Wolfman et al., 2003). However, thenodal, BMP4 and BMP7 pro-peptides do not act as extracellular

antagonists, but instead regulate mature ligand stability andprocessing, including ligand degradation in lysosomes, which limitsligand availability (Degnin et al., 2004; Dick et al., 2000; Le Goodet al., 2005). Similarly, the nodal pro-peptide associates with itsEGF-CFC family co-receptor Cripto in secretory vesicles near thecell surface (Blanchet et al., 2008). Cripto also forms complexeswith mature nodal and enhances signaling via the receptor kinasecomplex (see below) (Bianco et al., 2004). Recent evidencedemonstrates that the signaling Cripto-nodal-receptor complexenters a specialized endocytic pathway that is characterized by theprotein flotillin, possibly en route to its final degradation.Interestingly, many other TGF ligands are inactivated in theextracellular space by antagonists, such as noggin and chordin,which inhibit BMPs, and follistatin, which inhibits activins(Gazzerro and Canalis, 2006; Harrison et al., 2005). Theseextracellular antagonists help to establish the morphogen gradientsthat pattern early embryos, as discussed in an accompanying review(Umulis et al., 2009) (see Box 1).

The TGF receptor familyAll TGF ligands transmit biological information to cells by bindingto type I and type II receptors that form heterotetrameric complexesin the presence of the dimeric ligand (reviewed by Wrana et al.,2008). Five type II and seven type I receptors exist in humans andother mammals, and are characterized by a cytoplasmic kinasedomain that has strong serine/threonine kinase activity and weakertyrosine kinase activity, which classifies them as being dual-specificity kinases (Table 1) (reviewed by ten Dijke and Heldin,2006). The type I receptors are also known as activin receptor-likekinases (ALKs), a nomenclature that is employed to tackle theproblem of one ligand signaling via many receptors, or many ligandssignaling via the same receptor. TGF ligands also interact with co-receptors that either facilitate or limit receptor kinase signaling. Inaddition to the EGF-CFC/Cripto co-receptors discussed above, typeIII receptors, such as endoglin and the proteoglycan betaglycan(TGFR3; TRIII), regulate TGF signaling in mammals, as doesthe repulsive guidance molecule (RGM, also known as Dragon)family of co-receptors (reviewed by Wrana et al., 2008) (Table 1).

Ligand binding links the constitutively active type II receptorkinases to the dormant type I receptor kinases, allowing the type IIreceptor to phosphorylate the juxtamembrane part of thecytoplasmic domain of the type I receptor (Fig. 1), turning onreceptor kinase activity (reviewed by Wrana et al., 2008). Recentstructural analysis of TGF and BMP ligands bound to theirrespective type I and type II receptor ectodomains shows that TGFligands contact both receptors tightly, whereas the evolutionarilymore ancient BMPs associate more loosely with their receptors(Groppe et al., 2008). Binding of TGF to TRII (TGFR2) createsthe interface required for TRI (ALK5; TGFR1) type I receptorrecruitment to the complex.

D. melanogaster has five TGF family receptors, including thetype II receptors Punt (Put) and Wishful thinking (Wit), which bindthe BMP-like ligands Dpp, Gbb and Scw during fly developmentand which form complexes with the type I receptors Thickveins(Tkv) and Saxophone (Sax) (Table 1) (Affolter and Basler, 2007;Goold and Davis, 2007; Serpe et al., 2005). Put and Wit also pairwith the type I receptor Baboon (Babo) to mediate activin-likesignals from dActivin and Daw (Zhu et al., 2008). Theaccompanying review by Umulis et al. (Umulis et al., 2009)discusses how, in the developing wing disc, a gradient of BMP-likesignaling activity is achieved by the dual contribution of Dpp andGbb, which differentially bind to distinct receptor complexes.

REVIEW Development 136 (22)

Box 2. Role of TGF/BMP signaling in embryonic stemcellsStem cells exhibit self-renewing capacity and pluripotency ingenerating the multitude of embryonic and adult cell types of themetazoan body (reviewed by Rossi et al., 2008). Growth factors, suchas TGF and FGF, regulate stem cell self-renewal and differentiation.FGF2, the most widely used growth factor that supports mouse andhuman embryonic stem cell (ESC) self-renewal in culture, inducesTGF/activin ligands and receptors while suppressing BMP-likeactivities (Greber et al., 2007; Ogawa et al., 2007). Furthermore,pharmacological inhibitors of the TGF/nodal type I receptor familysuppress human and mouse ESC self-renewal (Ogawa et al., 2007).In general, TGF inhibits differentiation of pluripotent progenitorcells, whereas BMP induces their differentiation (Watabe andMiyazono, 2009) (Fig. 7A,B).

To promote self-renewal of ESCs, TGF/nodal signaling activatesSMAD2 and SMAD3, which directly induce Nanog, one of the crucialstem cell transcription factors (Xu, R. H. et al., 2008). TGF and FGFsignaling synergize by enhancing binding of Smad complexes to theNanog promoter. Interestingly, NANOG provides a molecular link forthe antagonism between TGF (the self-renewing factor) and BMP(the differentiation factor) in ESCs. NANOG binds to SMAD1,inhibiting its transcriptional activity and limiting the BMP signalingpotential that promotes early mesodermal differentiation or tissue-specific differentiation later in development (Suzuki et al., 2006). Thisexample is likely to be expanded to additional regulators of ESC self-renewal and differentiation as a result of genome-wide screens forthe transcription and signaling factors of these pathways (Chen etal., 2008).

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C. elegans has three TGF family receptors (Table 1) (Pattersonand Padgett, 2000). In the dauer pathway, DAF-7 signals via the typeII receptor DAF-4 and the type I receptor DAF-1. In the Sma/Mabpathway, which regulates body length, tail development and innateimmunity, DBL-1 signals via DAF-4 and the SMA-6 type I receptor.Which of these receptors mediate signals by UNC-129, TIG-2 andTIG-3 remains unknown.

Finally, T. adhaerens has one type II and three type I receptors(Huminiecki et al., 2009), which is compatible with a model inwhich the type II receptor represents the ligand-recognizing core,whereas the type I receptor is the downstream signaling effectorthat defines biological responses and that has diverged morerapidly to serve the new developmental processes of morecomplex organisms.

The Smad familyThe activated type I receptor phosphorylates cytoplasmic proteinsof the Smad family in their C-terminal regions (Figs 1 and 2). Smadsconsist of three domains: (1) an N-terminal Mad-homology 1 (MH1)domain that can interact with other proteins and carries nuclearlocalization signals (NLSs) and a DNA-binding domain; (2) amiddle linker domain that interacts with prolyl-isomerases andubiquitin ligases and that is enriched in prolines andphosphorylatable serines or threonines; and (3) a C-terminal MH2domain that binds to type I receptors and can interact with otherproteins, and that mediates Smad homo- and hetero-oligomerizationand mediates the transactivation potential of nuclear Smadcomplexes (Fig. 2) (reviewed by ten Dijke and Heldin, 2006). TheC-terminal phosphorylation of receptor-activated (R) Smads allows

Table 1. TGF pathways in humans, flies and worms

H. sapiens

Pathway BMP GDF Activin TGF AMH Inhibitors

Ligand BMP2, 4BMP5, 6, 7BMP8A, 8BBMP9, 10

GDF5, 6, 7GDF9b

GDF10, 11GDF15 (MIC1)------------------

GDF1, 3GDF8 (MYO)

GDF9

Inhibin AInhibin B

Nodal

TGF 1TGF 2TGF 3

AMH (MIS) BMP3Inhibin Inhibin CInhibin ELEFTYALEFTYB

RII BMPRIIActRIIA, ActRIIB

BMPRIIActRIIA, ActRIIB

ActRIIAActRIIB

T RII AMHRII N/A

RI BMPRIA (ALK3)BMPRIB (ALK6)

ALK2ALK1

BMPRIA (ALK3)BMPRIB (ALK6)

ALK2-------------------ActRIB (ALK4)

ALK7T RI (ALK5)

ActRIB (ALK4)ALK7

T RI (ALK5)--------------

ALK1ALK2

BMPRIA (ALK3)

BMPRIA (ALK3)BMPRIB (ALK6)

ALK2

N/A

RIII RGMa, b, c (+) Cripto 3 (+) Cripto 3 (–)Cripto 1 (+)

T RIII (+)Endoglin (+)Cripto 3 (–)

? T RIII (–)Cripto 3 (–)

R-Smad SMAD1, 5, 8 SMAD1, 5, 8–-----------------

SMAD2, 3

SMAD2, 3 SMAD2, 3------------------SMAD1, 5, 8

SMAD1, 5, 8 N/A

Co-Smad SMAD4 SMAD4 SMAD4 SMAD4 SMAD4 N/A

I-Smad SMAD6, 7 SMAD6, 7 SMAD7 SMAD7 SMAD6, 7 N/A

D. melanogaster C. elegans

Pathway BMP Activin Other Sma/Mab Dauer

Ligand DppGbbScw

dActivinDaw

MavMyo

DBL-1 DAF-7

RII PutWit

PutWit

? DAF-4 DAF-4

RI TkvSax

Babo ? SMA-6 DAF-1

RIII ? ? ? ? ?

R-Smad Mad dSmad2 ? SMA-2SMA-3

DAF-8DAF-14

Co-Smad Medea Medea ? SMA-4 DAF-3 (?)

I-Smad Dad ? ? TAG-68 (?) TAG-68 (?)Receptors are listed as type II (RII), type I (RI) and type III (RIII) co-receptors. Dashed lines separate groups of ligands or receptors based on the division into BMP andTGF /activin-like pathways. Ligands, type I receptors and R-Smads are color-coded: blue, BMP-like pathways; red, TGF /activin-like pathways. Question marks indicateunassigned signaling relationships. For two human ligands, GDF8 and GDF15, we provide their alternative names [myostatin (MYO) and macrophage inhibitory cytokine 1(MIC1)], as the latter are more commonly used in the literature. LEFTYA and B are also known as LEFTY2 and 1, respectively. The co-receptor T RIII is also known asbetaglycan. Cripto 1 and Cripto 3 are also known as TDGF1 and TDGF3, respectively. In the RIII group (+) or (–) indicates positive or negative effects, respectively, onsignaling by each co-receptor. N/A, not applicable.

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them to associate with the common-mediator (Co) Smad, SMAD4.The resulting Smad oligomer is thought to consist of a trimer of twoR-Smads and a single SMAD4 (such as a SMAD2-SMAD2-SMAD4 complex, a SMAD3-SMAD3-SMAD4 complex, or aSMAD2-SMAD3-SMAD4 complex), which is then shuttled into thenucleus. Nuclear Smad complexes bind to chromatin, and, togetherwith other transcription factors, regulate target gene expression (Fig.1) (reviewed by Massagué et al., 2005; Schmierer and Hill, 2007).TGF- or BMP-specific Smad complexes induce the expression ofthe inhibitory (I) Smads, SMAD6 and SMAD7 (Figs 1 and 2), whichnegatively regulate signaling strength and duration, thus forming anegative-feedback loop (reviewed by Itoh and ten Dijke, 2007).

All genomes sequenced to date possess the three fundamentalclasses of Smad proteins: R-, Co- and I-Smads (Table 1)(Huminiecki et al., 2009). In D. melanogaster, BMP-like signalingis mediated by a single R-Smad (Mad) and a single Co-Smad(Medea), despite the existence of two type I receptors (Tkv, Sax)(Affolter and Basler, 2007). In the activin-like pathways, the type Ireceptor Babo signals via dSmad2 (Smox) (an R-Smad) and Medea.A single I-Smad (Dad) also operates during wing imaginal discdevelopment (Tsuneizumi et al., 1997). Dad binds and inhibitssignaling from the Dpp/Scw/Gbb type I receptors Tkv and Sax, butnot from the dActivin type I receptor Babo (Kamiya et al., 2008).

In C. elegans, the Sma/Mab pathway engages two R-Smads,SMA-2 and SMA-3, that signal with a single Co-Smad, SMA-4(Patterson and Padgett, 2000). The dauer pathway has two R-Smads,DAF-8 and DAF-14, which have more divergent MH1 domains butconserved MH2 domains that suggest activation by thecorresponding type I receptors. The dauer pathway Co-Smad ispossibly DAF-3, which presents peculiar developmentalcharacteristics. Unlike the Co-Smads in other organisms and SMA-4 in C. elegans, the DAF-3 loss-of-function phenotype is distinctfrom those of loss-of-function mutations in the ligand, receptors orR-Smads of this pathway (Patterson et al., 1997). Furthermore, thereceptors and R-Smad seem to negatively regulate the function ofDAF-3. Thus, DAF-3 is classified as a Co-Smad only on the basisof sequence similarity to other Co-Smads (Huminiecki et al., 2009).Finally, TAG-68 is classified as an I-Smad based on phylogeneticarguments, although functional evidence for such a role is currentlyabsent (Savage-Dunn et al., 2003). T. adhaerens also has threedistinct Smad classes, which suggests that the three differentfunctional features of Smad proteins evolved early during metazoanevolution (Huminiecki et al., 2009).

A fundamental feature of all TGF signaling pathways is theirdivision into TGF-like and BMP-like cascades, a classification basedon the specificity of interaction between the so-called L45 loop of the


RI

P

RII

TGFββ

Co-Smad

Smad3

Latent TGFβ

PSmad2

PTight

junction TAK1

JNK

Cytoplasm

Nucleus

RI

P

RII

BMP

Smad1P

Smad5P

RI

P

RII

TGFβ

GSK3βRho

Par6 P

Smurf

Actin

TRAF6

p38

ShcAP

ERK

Co-Smad

Other substrates

Src

Smad7

mRNA

TF

R-Smad PCo-Smad

Chromatin

Smad7

Noggin/BMP

A BC

Smad6/7

Smad8P

mRNA

TFChromatin

P

Rho

?

Actin

Stress fibersFocal adhesionsMotility

ActivinNodal

RI

P

RII

Follistatin/Activin

Smad7

Smad6ROCK

LIMK2

?

ChromatinmRNA

TF

R-Smad PCo-SmadR-Smad

P

R-SmadP

Fig. 1. TGF and BMP signaling. (A,B)The (A) TGF and activin/nodal and (B) BMP pathways, with their corresponding Smad proteins andmechanisms of inhibition by I-Smads (Smad6/7). The latent TGF complex and the extracellular antagonists, follistatin (bound to activin) and noggin(bound to BMP) are shown. (C)Non-Smad signaling pathways downstream of the TGF receptors [RI, TRI (ALK5) and RII, TRII (TGFR2)]. Thenuclear Smad complexes that lead to gene regulation are shown for each pathway. In these complexes, the Smad trimer most likely contains two R-Smad (identical or different) and one Co-Smad subunit. In addition to the major signaling pathways shown, TGF also activates BMP R-Smads incertain contexts (see text). BMP (bone morphogenetic protein), Erk (extracellular signal-regulated kinase), LIMK2 (LIM domain kinase 2), JNK (Jun N-terminal kinase), p38 (p38 MAPK), PAR6 (partitioning-defective 6 homolog), Rho (Ras homolog), ROCK (Rho-associated, coiled-coil-containingprotein kinase), SHCA (SH2 domain-containing sequence A), Smurf (Smad ubiquitylation regulatory factor), Src (Rous sarcoma virus oncoprotein),TGF (transforming growth factor ), TAK1 (TGF-activated kinase 1), TF (transcription factor), TRAF6 (tumor necrosis factor receptor-associatedfactor 6).

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type I receptors and the L3 loop of the MH2 domains of R-Smads(Fig. 2) (reviewed by ten Dijke and Heldin, 2006). TGF/activinpathways signal via SMAD2, SMAD3, and BMP/GDF pathways viaSMAD1, SMAD5 and SMAD8. However, in a diversity of cell typesin culture, such as endothelial, immortalized epithelial, adenoma andcarcinoma cell lines and even NIH-3T3 fibroblasts and chondrocytes(Daly et al., 2008; Finnson et al., 2008; Goumans et al., 2003; Liu etal., 2008), TGF signaling can also activate SMAD1 and SMAD5.Originally, TGF was shown to bind to two type I receptors, thusactivating SMAD2 and SMAD3 via TRI, and SMAD1, SMAD5 andSMAD8 via the BMP type 1 receptor ALK1 (ACVRL1). ALK1 isexpressed mainly in endothelial cells, where SMAD2, SMAD3signaling inhibits and SMAD1, SMAD5, SMAD8 signaling promotesproliferation and migration (Goumans et al., 2003). However, TGFcan also activate SMAD1 and SMAD5 via two additionalmechanisms. First, in immortalized EpH4 mouse mammary epithelialcells and in MDA-MB-231 human mammary carcinoma cells, TGFinduces SMAD1 and SMAD5 C-terminal phosphorylation viaheteromeric receptor complexes that form between TRII and TRI,as well as between TRII and the BMP type I receptors ALK2(ACVR1) and BMPRIA (ALK3) (Daly et al., 2008). Second, in 4T1

mouse mammary carcinoma cells, TGF leads to SMAD1 andSMAD5 phosphorylation without the requirement of a BMP-like typeI receptor because the TRI receptor kinase can directly phosphorylateSMAD1/5 (Liu et al., 2008). These findings suggest that a re-evaluation of TGF family signaling is needed through the elucidationof the type I receptors and Smad pathways that function in specificphysiological and developmental contexts.

TGF family signaling via non-Smad signalingproteinsOther proteins mediate TGF signaling in addition to Smads (seealso Moustakas and Heldin, 2005), and here we describe themechanistically best-established examples (Fig. 1C). TRIIphosphorylates the polarity protein PAR6, which regulates the localdegradation of the RHOA small GTPase that controls the assemblyof intercellular tight junctions in mammalian cells (Ozdamar et al.,2005). As tight junctions disassemble, epithelial architecturedisintegrates, followed by de-differentiation known as the epithelial-to-mesenchymal transition (EMT), an important developmental anddisease-associated process that is regulated by TGF signaling(reviewed by Moustakas and Heldin, 2007). During EMT, in

S*

(S*)

T*

(T*)

(T*)

(T*)

(T*)

T*

(T*)

T*

S*

(S*)

S*S*

S*

SMAD2

SMAD3

SMAD1

SMAD5

SMAD8

e3

β-hNLS

SMAD4 β-hNLS NES

L3

SAD

SMAD6

SMAD7NLS ?

NES ? L3

β-hNLS

β-hNLS NES

L3

β-hNLS

β-hNLS

S*MS*

S*VS*

S*VS*

S*VS*

S*VS*

NES

UbSumo

Ac

S* S*

S* S*

(S*)

(S*)

(S*)

(S*)

R-Smads

Co-Smad

I-Smads NLS ?

NES ? L3

Ac

Ac

PY

PY

PY

PY

PY

PY

PY

Me

(S*)

S*S*T*

(S* S*T*S*)

T*

(S* S*T*S*)

A

B

C

L3

L3

L3

L3

Fig. 2. The Smad family. Simplified structures of the eight human Smad proteins divided into (A) Receptor-activated (R) Smads; (B) common-mediator (Co) Smad; and (C) inhibitory (I) Smads. The conserved N-terminal Mad-homology 1 (MH1) (blue) and C-terminal MH2 (green) domainsare shown. Highlighted are the nuclear localization signal (NLS, striped box); the two unique inserts of SMAD2 (triangles), the second of whichcorresponds to exon 3 (e3); the -hairpin domain that binds to DNA (-h, black box); the proline-tyrosine (PY) motif in the linker domain that isrecognized by the WW domain of Smurf family proteins; the Smad activation domain (SAD) at the linker-MH2 border; the nuclear export signal(NES, hatched box); and the L3 loop of the MH2 domain. Phosphorylatable serine and threonine residues are shown; S/T* indicates experimentallyproven phosphorylation sites; (S/T*) indicates a conserved residue with a predicted phosphorylation motif that awaits experimental validation. TheC-terminal serines that are phosphorylated by the type I receptor kinases are shown in green (S*VS*, S*MS*); red S/T residues are phosphorylatedby the MAPKs ERK1/2; brown S/T residues are phosphorylated by protein kinase C and by calmodulin-dependent kinase II; blue S/T residues arephosphorylated by cyclin-dependent kinases; and black S/T residues are phosphorylated by glycogen synthase kinase 3. Sumoylation (Sumo),ubiquitylation (Ub), methylation (Me) and acetylation (Ac) sites are indicated with colored arrowheads.

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addition to tight junctions, adherens junctions and desmosomes ofpolarized epithelial cells are destroyed and remodeled to give rise tomesenchymal-like cells that are motile and invasive. It should benoted that, in addition to the above direct mechanism of tightjunction disassembly, TGF elicits EMT via Smad signaling,leading to the transcriptional induction of major inducers of thisdifferentiation process (Thuault et al., 2008).

Whereas the TGF-PAR6 pathway locally degrades RHOA in abreast epithelial cell culture model, other studies have demonstratedthe positive activation of Rho GTPase signaling by TGF and BMPreceptors in diverse cell types (reviewed by Kardassis et al., 2009).However, the mechanism of Rho activation by TGF receptorsremains unclear (Fig. 1C).

In a distinct mechanism, the TGF type I receptor phosphorylatesboth serine and tyrosine residues in the SHCA (SHC1) adaptor,which then recruits the adaptor protein GRB2 and the Ras guanineexchange factor (GEF) son of sevenless (SOS) in mammalian cells(Fig. 1C) (Lee, M. K. et al., 2007). This leads to activation of theRas-Raf-MEK-Erk mitogen-activated protein kinase (MAPK)signaling cascade, which can regulate cell proliferation or migration.Future work might decipher to what extent a specific biologicalresponse to TGF receptor signaling depends on its strongserine/threonine, or on its weaker tyrosine kinase, activity.

The tyrosine kinase Src can phosphorylate Tyr284 in thecytoplasmic domain of the TRII receptor, leading to GRB2 andSHC recruitment and to the activation of the p38 MAPK pathway(Galliher and Schiemann, 2007). Src-dependent TRIIphosphorylation regulates breast cancer cell proliferation andinvasiveness, possibly without affecting the Smad signaling output(Galliher-Beckley and Schiemann, 2008).

As a final example, TGF-induced receptor heterotetramersrecruit the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) to the TRI cytoplasmic domain inmammalian cells (Fig. 1C). TRAF6 ubiquitylates and activates thecatalytic activity of the TGF-activated kinase 1 (TAK1; MAP3K7),leading to activation of the p38 and c-Jun N-terminal kinase (JNK)cascades, which regulate apoptosis or cell migration (Sorrentino etal., 2008; Yamashita et al., 2008). The TRI kinase activity isdispensable for this pathway (Sorrentino et al., 2008).

The developmental significance of these non-Smad pathwaysremains to be elucidated. However, it has recently been shown thatboth p38 MAPK and Smad signaling play important rolesdownstream of TGF during mouse palate and tooth development(Xu, X. et al., 2008). In Xenopus, the adaptor protein TRAF4 haspositive signaling roles in mediating both the BMP and nodal signalsthat regulate neural crest differentiation and migration (Kalkan et al.,2009). TRAF4 is a substrate of the ubiquitin ligase Smadubiquitylation regulatory factor 1 (SMURF1), which polyubiquity-lates and promotes TRAF4 degradation, thus limiting the activity ofthe BMP and nodal pathways in the Xenopus neural plate. Signalingvia multiple effectors enables the TGF pathways to be controlled byother pathways, as we discuss below, through the mechanisms thatcontrol Smad function in different cell compartments.

TGF receptor regulation and endocytosisReceptor phosphorylation is important for TGF family signaltransduction, and thus receptor dephosphorylation might also beimportant. New evidence shows that TGF/nodal receptors arereciprocally regulated by B and B, two isoforms of regulatorysubunit B of the protein phosphatase 2A (PP2A). PP2A that containsthe B subunit positively, whereas PP2A that contains the Bsubunit negatively, regulates receptor signaling in Xenopus embryos

and in mammalian cells (Batut et al., 2008). The direct moleculartargets of PP2A and the serine or threonine residues that theydephosphorylate await further analysis.

In addition, TRI can be sumoylated by an as yet unknown sumo-ligase in mammalian cells (Fig. 3A), which enhances TGFsignaling by facilitating SMAD3 recruitment to the receptor forphosphorylation (Kang et al., 2008). TRI sumoylation mightprovide a docking site for an adaptor that mediates SMAD3 bindingto the receptor, might induce a conformational change in the receptorthat is required for SMAD3 binding, or might be coupled to theinternalization mechanism.

The activated TGF receptors are internalized via clathrin-coatedpits into early endosomes, where the receptors encounter Smadanchor for receptor activation (SARA; ZFYVE9), which facilitatesthe recruitment of SMAD2 and SMAD3 to TRI and theirsubsequent phosphorylation (Fig. 3A,B) (Tsukazaki et al., 1998).SARA binds SMAD2 and SMAD3, but not BMP R-Smads, and alsocontacts the TRI receptor. A homolog of SARA, endofin(ZFYVE16), plays a similar role in BMP pathways (Fig. 3D,E) (Shiet al., 2007). However, endofin can also operate in TGF pathwaysby scaffolding SMAD4 and by mediating the formation ofcomplexes of SMAD2, SMAD3 and SMAD4 in association with thetype I receptor (Chen et al., 2007).

In mammals, SARA cooperates with the cytoplasmicpromyelocytic leukemia (cPML) tumor suppressor protein, whichstabilizes the SARA-Smad complex (Fig. 3B) (Lin et al., 2004). Thisprocess also involves another adaptor, the PML competitor for TGIFassociation (PCTA), which binds to the nuclear homeodomainrepressor protein TGIF (5�TG3�-interacting factor) and retainscPML in the nucleus (Faresse et al., 2008). In response to TGF,PCTA releases cPML to translocate to the cytoplasm, reach SARAand promote TGF signaling.

The Drosophila ortholog, dSARA, plays a similar role tomammalian SARA during Dpp/Mad signaling (Bökel et al., 2006).Since dSARA has no other homolog in Drosophila, it might mediateboth Mad and dSmad2 signaling, although this remains to beestablished. In the epithelial cells of the Drosophila wing, whichundergo apical and basolateral differentiation, Dpp receptor-dSARAcomplexes reside in apically located endosomes and are preciselysegregated during cell division (Bökel et al., 2006). In this way, thedaughter cells receive equal numbers of signaling complexes, whichensures the conservation of signaling strength from mother todaughter cells.

Many additional cytoplasmic regulators of early TGF receptorsignaling have been described recently (Fig. 3B). Most notably,the small GTPase RAP2 inhibits activin/nodal receptor recycling,thus controlling receptor levels on the surface of Xenopusembryonic cells (Choi et al., 2008). During signaling, RAP2antagonizes the negative effects of SMAD7, thus positivelycontributing to nodal signal propagation and the onset ofgastrulation. In addition, RIN1, a RAB5 GEF, promotes TGFreceptor endocytosis and overall signaling, which contributes tothe pro-tumorigenic action of TGF in breast epithelial cells (Huet al., 2008). The PDZ-containing protein erbin (ERBB2IP) bindsto phosphorylated SMAD2, SMAD3, prevents their associationwith SMAD4 in mammalian cells, and produces opposite effectson signaling to SARA or endofin (Dai, F. et al., 2007). The proteinDapper 2 (DACT2) contributes to TGF receptordownregulation, which modulates nodal signaling in Xenopus,zebrafish, and mice (Su et al., 2007), although its partners andmechanism of action require further exploration. It would beinteresting to elucidate the mechanism that regulates the


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recruitment of positive regulators, such as SARA, endofin andRIN1, and of negative regulators, such as RAP2, erbin andDapper 2, to the TGF receptor complex to ensure appropriate R-Smad phosphorylation and SMAD4 association.

Receptor endocytosis both controls the flow of signaling andregulates the availability of TGF ligand on the cell surface. An invitro kinetic analysis of TGF ligand bioavailability has shown thatconstitutive endocytosis of TRII depletes excess ligand (Clarke etal., 2009). This mechanism enables a cell to fine-tune the level ofsignaling growth factor on the cell surface.

The regulatory proteins described above highlight the linkbetween TGF signaling and the regulation of receptorinternalization. However, the developmental relevance of many ofthese factors awaits further analysis.

TGF receptor downregulation and the role of I-SmadsTGF ligand-receptor complexes can be additionally internalizedvia lipid rafts into caveolae, and then into lysosomes, where theligand-receptor complex is degraded (Di Guglielmo et al., 2003)(Fig. 3C). TGF receptor trafficking via caveolae is marked by theirassociation with I-Smads and SMURF1 or SMURF2 ubiquitinligases, which negatively regulate the signaling cascade.

The inhibitory Smads, SMAD6 and SMAD7, bind to type Ireceptors, thereby competitively inhibiting R-Smadphosphorylation and recruiting phosphatases and Smurf ubiquitinligases to downregulate receptor levels and function (reviewed byItoh and ten Dijke, 2007). Whereas SMAD7 inhibits both TGFand BMP pathways, SMAD6 more selectively inhibits BMPpathways (Fig. 1) and shows greater selectivity for the BMP typeI receptors ALK1, ALK2, ALK3 and ALK6, as demonstratedrecently in mammalian cells. Furthermore, SMAD6 binds witheven higher affinity to specific amino acid residues in theBMPRIA (ALK3) and BMPRIB (ALK6) kinase domains, than toALK1 and ALK2 domains (Goto et al., 2007). By contrast,SMAD7 shows broader specificity as it binds to all type Ireceptors via specific lysine residues in its MH2 domain(Mochizuki et al., 2004).

Two regulatory mechanisms that mediate the SMAD7-dependent ubiquitylation and downregulation of the TGFreceptor have recently been uncovered in mammalian cells (Fig.3C). The chaperone protein HSP90 binds to TRII and to TRIand protects them from ubiquitylation by SMURF2, positivelycontributing to TGF signaling (Wrighton et al., 2008).Conversely, the AMP-regulated kinase member salt-induciblekinase (SIK) is induced at the mRNA and protein levels by TGF

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Fig. 3. TGF receptor endocytosis and downregulation. (A,D)The (A) TGF and (D) BMP receptor complexes are shown at the plasmamembrane. Type II receptors are labeled as RII, type I receptors as RI. Sumoylation (S) of the TRI that positively influences Smad signaling ishighlighted. (B)TGF receptors are internalized in endosomes characterized by the presence of the endocytic protein SARA and the regulatoryadaptor cPML. The receptors can signal by SMAD2 and SMAD3 phosphorylation and activation of the Co-Smad and by accumulation of nuclearSmad complexes on chromatin. Key regulatory proteins involved in the process of receptor endocytosis are listed on the side of the arrow thatindicates the flow of endosomal trafficking. (C)The TGF receptor degradation pathway via caveolae, which are characterized by the presence ofcaveolin 1 (CAV1), and the key regulatory proteins involved as shown within the box. (E)The corresponding endocytic pathway for BMP receptorswith the endocytic protein endofin is less well understood. (F)The corresponding caveolae-based degradation pathway for the BMP receptorsremains unexplored (?). Key established regulatory proteins are shown in the box. BMP (bone morphogenetic protein), cPML (cytoplasmicpromyelocytic leukemia protein), HSP90 (heat-shock protein of 90 kDa), RAP2 (Ras-related protein 2), RIN (Ras-like protein expressed in neurons),SARA (Smad anchor for receptor activation), SIK (salt-inducible kinase), Smurf (Smad ubiquitylation regulatory factor), TGF (transforming growthfactor ).

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signaling, concomitantly with the induction of SMAD7 andSMURF2 (Kowanetz et al., 2008). SIK binds to SMAD7 and toTRI to promote receptor downregulation. The C. elegans SIKortholog, KIN-29, exhibits a conserved function by regulatingbody size in the Sma/Mab pathway; however, the molecularmechanism of KIN-29 action in worms remains unexplored(Maduzia et al., 2005).

Although SMAD7 primarily acts at the type I receptor level, italso resides in the nucleus, and new evidence suggests that it canbind to DNA and to nuclear complexes of SMAD2, SMAD3 andSMAD4, disrupting their complexes and inhibiting theirtranscriptional activity (Fig. 4) (Zhang et al., 2007).

Based on the importance of the mechanisms of TGF receptorendocytosis and downregulation, and the links of such mechanismsto the nucleocytoplasmic shuttling of Smads (see below), futurestudies into the biology of I-Smads promise to reveal interesting andnovel findings.

Regulation of Smad trafficking by motor proteinsIn parallel to TGF receptor endocytosis, R-Smads becomephosphorylated by the type I receptors and accumulate in thenucleus. However, Smads, like the receptors, show dynamicmobility and shuttle in and out of the nucleus even when they arenot activated by receptors (reviewed by Moustakas and Heldin,2008). The cytoplasmic trafficking of both TGF receptors andSmads is often mediated by motor proteins that are associatedwith microtubules. Motor proteins are important both before andafter R-Smad C-terminal phosphorylation.

Accordingly, Smads interact with kinesin 1, which mediates therecruitment of SMAD2 to the receptor complex in Xenopus andmammalian cells (Batut et al., 2007). Smads are sequestered awayfrom the receptors when bound to microtubules, from where theycan be released, for example, by connexin 43 (GJ1), whichcompetes with microtubules to bind Smads (Dai, P. et al., 2007).Additional motor proteins, such as the dynein light chain km23-1

(DYNLRB1), also promote Smad traffic towards the nucleus ofmammalian cells (Jin et al., 2007). The developmental roles of theconnexin 43 and km23-1 mechanisms have not yet beenspecifically addressed.

Microtubules also transport specialized pools of Smad proteins.For example, the pool of SMAD1 that has been subjected toinhibitory phosphorylation in its linker domain by Erk MAPKs(see below) moves towards the centrosome via microtubules,where it is degraded by proteasomes (Fuentealba et al., 2008).Interestingly, when mammalian embryonic and adult cells, orDrosophila blastoderm cells, complete mitosis, phosphorylatedSMAD1 and the centrosomal degrading apparatus segregate toonly one of the two daughter cells (Fuentealba et al., 2008). Thus,Smad trafficking and segregation to daughter cells is regulateddevelopmentally in a stage-specific manner. Additional studies inXenopus embryos show that SMAD2-SMAD4 complexes arerecruited to chromatin during every cell division, when mitosisdissolves the nuclear envelope (Saka et al., 2007). Again, thisevent is regulated developmentally as it does not occur prior to themid-blastula transition. Moreover, SMAD3 regulates the activityof the anaphase-promoting complex, a primary initiator of mitosisduring the mammalian cell cycle (Fujita et al., 2008). Whetherthese three mechanisms of Smad regulation during embryonic cellmitosis constitute one and the same process remains to beelucidated.

Mechanisms of Smad shuttling through thenuclear envelopeSmad nuclear import via nuclear pores is mediated bynucleoporins, which are integral constituents of the pores, and byimportins, carrier proteins that bind to both cargo proteins andnucleoporins and that catalyze their nuclear translocation in anenergy-dependent manner (reviewed by Moustakas and Heldin,2008). All Smads have a conserved NLS in their MH1 domain(Fig. 2), which binds to specific importins, such as importin (in


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Fig. 4. Regulation of the nuclear Smadcomplex. Following TGF receptor activation, theactivated Smad complex translocates to thenucleus and associates with various Smad partners[represented by the blue, triangular transcriptionfactor (TF) and by examples discussed in the text]and undergoes post-translational modifications (A,acetylation; U, ubiquitylation; S, sumoylation; P,phosphorylation). Black arrows indicate a positiveeffect of a post-translational modification on Smadtranscriptional function, red T-bars indicate anegative effect. (Left) Derepression of Smad targetgenes when the SNON/SKI co-repressors areproteasomally degraded after being ubiquitylatedby the ubiquitin ligases arkadia, SMURF2 orAPC/CDH1. APC/CDH1 (anaphase-promotingcomplex and its ubiquitin ligase subunit CDH1),BRG1 (Brahma-related gene 1), ETS1 (v-etserythroblastosis virus E26 oncogene homolog 1),HHM (human homolog of Maid), IKK (IB kinase), SMURF2 (Smad ubiquitylation regulatory factor2), TFAP2A (transcription factor activatingenhancer-binding protein 2), TGF (transforminggrowth factor ).

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the case of SMAD1 and SMAD3) and importin (in the case ofSMAD4). A recent genome-wide study in Drosophila S2 cellsreported the roles of additional importins, such as Msk, whichimports Mad, and of its mammalian orthologs, importin 7 andimportin 8, which import SMAD2, SMAD3 and SMAD4 (Xu etal., 2007; Yao et al., 2008).

Smad proteins have characterized nuclear export signals(NESs) in their MH2 (SMAD1, SMAD3) or linker (SMAD4)domains (Fig. 2). The NESs bind specific exportins – exportin 4for SMAD3 and exportin 1 for SMAD1 and SMAD4 – and exportis catalyzed by the small GTPase RAN. Recently, a new exportinwas described for SMAD2 and SMAD3, the RAN-bindingprotein 3 (RANBP3), which is a known exportin family member(Dai et al., 2009). RANBP3 was shown to recognizedephosphorylated nuclear SMAD2 and SMAD3 proteins andexport them to the cytoplasm. Importins and exportins forSMAD5 and SMAD8 or for the I-Smads remain to becharacterized. However, as we discuss below, our understandingof how Smads are transported into the nucleus has beensignificantly advanced by recent findings.

Regulation of Smad nuclear shuttlingThe dynamic movement of Smads in and out of the nucleus is highlyregulated. A recent mathematical model of SMAD2 and SMAD4trafficking reported that their nuclear accumulation in response toTGF reflects a shift in the equilibrium between the cytoplasmic andnuclear pools of Smads that is brought about by a decrease in nuclearexport (Schmierer et al., 2008). During signaling, however, low-level R-Smad dephosphorylation by nuclear phosphatases, amongother factors, continues to ensure their subsequent nuclear export(Schmierer et al., 2008).

R-Smad shuttling is regulated not only by cycles of receptor-mediated C-terminal phosphorylation and dephosphorylation bynuclear phosphatases (reviewed by Wrighton et al., 2009), butalso by sumoylation and ubiquitylation. Sumoylation of SMAD3by the protein inhibitor of activated Stat y (PIASy; PIAS4) sumo-ligase promotes its nuclear export in mammalian cells (Imoto etal., 2008). Sumoylation of Medea, by an as yet unidentified sumo-ligase, also promotes its nuclear export, providing negativeregulation that restricts the competence of early Drosophilaembryonic cells to respond to Dpp (Miles et al., 2008). Thismechanism resembles the previously established role ofmammalian SMAD4 sumoylation by PIAS ligases (reviewed byLönn et al., 2009). It is possible that in vivo Smad sumoylationmight not negatively regulate TGF signaling; rather, it mightpromote continuous Smad shuttling. However, underoverexpression conditions, sumo-ligases may shift the shuttlingequilibrium by pushing Smads to the cytoplasm, thus reducingtheir time in the nucleus.

SMAD4 can also be monoubiquitylated (Morén et al., 2003) bythe nuclear ubiquitin ligase TIF1 (ectodermin; TRIM33), whichpromotes its nuclear export and inhibits the formation of nuclearcomplexes of SMAD2, SMAD3 and SMAD4 (Dupont et al., 2009).Once monoubiquitylated, SMAD4 is exported from the nucleus(Wang et al., 2008), whereupon fat facets in mouse (FAM; USP9X)deubiquitylates it, recharging it for subsequent cycles of shuttling,as demonstrated in Drosophila, Xenopus and human cells (Dupontet al., 2009).

Other mechanisms also regulate the nuclear residence andfunction of Smad complexes. Heteromeric complexes of SMAD2,SMAD3 and SMAD4 bind to the shuttling protein transcriptionalco-activator with PDZ-binding motif (TAZ) in the nucleus of

human cells, and are then recruited to chromatin via factors suchas the activator-recruited co-factor (ARC) protein ARC105(MED15), a member of the Mediator complex that ensures theprogression of gene transcription (Varelas et al., 2008). TAZ isregulated by phosphorylation and by interaction with 14-3-3family adaptors that control its timely residence in the nucleus.Furthermore, the Drosophila nuclear lamin Otefin interacts withMedea and tethers Smad complexes to the nuclear envelope(Jiang et al., 2008). The Otefin-Medea complexes bind to specificgene-regulatory elements that control germline stem celldevelopment (Jiang et al., 2008). Future research into Smadshuttling and the regulation of Smad compartmentalization willbring to light additional regulatory mechanisms of TGFsignaling.

Negative regulation of Smad signaling byphosphorylation and ubiquitylationIn addition to regulating Smad nucleocytoplasmic shuttling, C-terminal tail dephosphorylation, linker domain phosphorylation andubiquitylation are implicated in the negative regulation of Smadsignaling (Figs 2 and 4).

The developmental importance of such post-translationalmodifications has been recognized during BMP-dependentneurogenesis in Xenopus and mouse C2C12 osteoblastdifferentiation. Fibroblast growth factor (FGF) signaling via Ras-Erk MAPK negatively regulates BMP signaling, as Erk (and GSK3kinase) directly phosphorylates the SMAD1 linker, leading torecruitment of SMURF1 and to the proteasomal degradation ofSMAD1 in perinuclear centrosomes (Fuentealba et al., 2007;Sapkota et al., 2007). During Xenopus neurogenesis, SMAD1degradation is triggered in three ways: by chordin antagonisingBMP activity extracellularly; by the Wnt antagonist Dickkopf 1blocking Wnt activity extracellularly; and by Erk MAPK pathwayactivation via FGF and insulin-like growth factor (IGF) signaling.Thus, FGF/IGF signaling provides negative feedback to BMPsignaling in the developing nervous system. Conversely, Wntsignaling induces GSK3 degradation, leading to decreasedSMAD1 linker phosphorylation and to its prolonged signaling, anevent that is required for Xenopus epidermal differentiation(Fuentealba et al., 2007). This is a good example of positive cross-talk between Wnt and BMP signaling during skin differentiation.

GSK3 also negatively controls TGF signaling, as it directlyphosphorylates SMAD3 in its MH1 domain in mammalian cells(Guo et al., 2008a). This phosphorylation is followed by theubiquitylation and proteasomal degradation of SMAD3, whichregulate its steady-state levels. By contrast, upon TGF receptorphosphorylation, SMAD3 can be further phosphorylated in its MH2domain by casein kinase 1 2, which leads to the specificubiquitylation and degradation of the activated form of SMAD3(Guo et al., 2008b).

A recent report has shed more light on the complexity of Smadregulation through the phosphorylation of its linker domain (Wanget al., 2009). After SMAD3 C-terminal phosphorylation by TRI inmammalian cells, nuclear GSK3 and cyclin-dependent kinasesphosphorylate three distinct SMAD3 linker residues,downregulating its transcriptional activity. Thus, TGF signalingtightly controls the activity of one of its main transducers throughhighly regulated phosphorylation events.

Recent evidence also shows that during mitosis of mammaliancells in culture, the kinase MPS1 (TTK) can directly C-terminallyphosphorylate SMAD2 and SMAD3, thus activating their nuclearactivities in the absence of TGF receptor activation (Zhu et al., D

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2007). This is one of the first clear TGF-independent mechanismsthat engages Smads and mimics the action of TGF. Thus, whereasR-Smad C-terminal phosphorylation by type I receptor kinases is apositive regulator of TGF family signaling, Smad phosphorylationby other kinases can negatively affect TGF family signaling in acell cycle-, developmental- or tissue-specific manner.

Transcriptional regulation by SmadsThe list of transcription factors to which Smads bind to regulate geneexpression continues to grow (see Table S1 in the supplementarymaterial) (reviewed by Feng and Derynck, 2005). Nuclear Smadcomplexes bind with weak affinity to Smad-binding elements(SBEs) on DNA (reviewed by Schmierer and Hill, 2007). Notably,the most common isoform of SMAD2 fails to bind to SBEs owingto an insertion within its DNA-binding domain, which resides in theMH1 domain of all Smads (see Fig. 2). SMAD3 recognizes 5�-GTCTG-3� as its SBE. By contrast, the BMP Smads and SMAD4recognize GC-rich sequences that have less conserved motifs, whichare sometimes in close proximity to an SBE. In general, recruitmentof Smad complexes to chromatin is dependent on their directinteraction with transcription factors that bind to DNA with higheraffinity (Fig. 4).

Upon binding to DNA and to their transcriptional partners, Smadsrecruit co-activators and histone acetyltransferases, such as p300,C/EBP-binding protein (CBP) and p300/CBP-associated factor(P/CAF), facilitating the initiation of transcription (reviewed bySchmierer and Hill, 2007). Recent evidence has shown thatp300/CBP also acetylates SMAD2/3, enhancing their DNA-bindingactivity in mammalian cells (Simonsson et al., 2006; Tu and Luo,2007). Conversely, histone deacetylases inhibit SMAD1transcriptional activity during neuronal differentiation in the mouseembryonic brain (Shakéd et al., 2008). However, direct acetylationor deacetylation of BMP-specific Smads has yet to be demonstrated.

The negative regulation of Smad signaling by Smadubiquitylation was summarized above. Positive regulation of nuclearSmad signaling by ubiquitylation has more recently come to lightfrom studies in mouse embryos and in mammalian cells (Mavrakiset al., 2007). Nuclear Smad complexes associate with the ubiquitinligase arkadia (RNF111) in a ligand-dependent manner and promotethe ubiquitylation and degradation of their interacting co-repressorsSKI and SNON (SKIL) (Le Scolan et al., 2008; Levy et al., 2007;Nagano et al., 2007). This mechanism brings about the derepressionand transcriptional induction of target genes by nuclear Smads (Fig.4). Interestingly, the proteasomal degradation of SNON depends onits phosphorylation by TAK1, the non-Smad effector of TGFsignaling (see Fig. 1C) (Kajino et al., 2007). However, it is unclearwhether SNON ubiquitylation by arkadia requires its priorphosphorylation by TAK1. Arkadia also ubiquitylates the inhibitorySMAD7 (Koinuma et al., 2003), but whether this process takes placein the nucleus or in the cytoplasm awaits clarification.

Genome-wide screens have revealed an association betweenSmads and the SWI/SNF family chromatin remodeling proteinBrahma-related gene 1 (BRG1; SMARCA4) and the DNA-bindingproteins ETS1 and transcription factor activating enhancer-bindingprotein 2 (TFAP2) (Koinuma et al., 2009; Xi et al., 2008). Acurrent model suggests that chromatin-bound Smads cannot performtranscriptional work in the absence of essential chromatinremodeling factors, such as BRG1 and the mediator componentARC105 (reviewed by Schmierer and Hill, 2007). Interestingly,ARC105 localization in distinct chromatin domains is regulated byTAZ, the nuclear Smad-tethering factor (Varelas et al., 2008). Theseearly reports open the door to future studies that might demonstrate

how TGF alters the dynamic architecture of chromatin, leading togene-specific transcriptional induction or repression. Such researchmight, for the first time, establish links between the epigeneticregulation of chromatin and the function of the TGF pathways.

Regulatory mechanisms of Smad transcriptionalco-factorsFrom the numerous Smad-transcription factor complexes and theirresulting mechanisms of target gene regulation (see Table S1 in thesupplementary material) (Feng and Derynck, 2005), we highlighthere a few selected examples that are of demonstrated or potentialdevelopmental relevance.

Xenopus mesoderm specification is driven by the concerted actionof TGF/activin and FGF-Ras-Erk MAPK signaling (Cordenonsi etal., 2007). The FGF-Ras-Erk MAPK pathway acts in distinct regionsof the developing Xenopus embryo, such as in the marginal zone,and induces, via phosphorylation, the activity of casein kinases,which then phosphorylate serines 6 and 9 of the tumor suppressorp53, contributing to mesoderm development. This phosphorylationactivates the transcriptional activity of p53, making it competent topair with Smads. This interaction leads to the transcriptionalinduction of mesoderm-defining genes, such as the transcriptionfactors Snail, Xbra (brachyury) and Mix.2. Conversely, duringXenopus ectoderm specification, p53 is inhibited by the zinc-fingerprotein XFDL156 (Sasai et al., 2008). This mechanism is essentialfor preventing the aberrant activation of nodal signaling in theectoderm, the developmental fate of which depends primarily on theactivity of BMP pathways. Although the above example emphasizespositive cross-talk between FGF and activin signaling duringXenopus mesoderm specification, this should not be interpreted asthe only developmental FGF signaling mechanism during frogmesoderm induction. The FGF response is multifactorial andmultigenic, as revealed by recent genome-wide transcriptomicscreens (Branney et al., 2009). Interestingly, although Smadscooperate with wild-type p53 to promote developmental processes,they also cooperate with mutant p53, which often accumulates inhuman cancers (Adorno et al., 2009; Kalo et al., 2007). The Smad-mutant p53 complex represses TGFBR2 transcription, leading to theinduction of pro-metastatic genes.

An unexpected transcriptional partner of SMAD2 and SMAD3 isthe well-characterized IB kinase (IKK; CHUK), whichparticipates in the nuclear factor B (NFB) pathway (Descargueset al., 2008). In the epidermis of Smad4-null mice, a complex ofSMAD2, SMAD3 and IKK forms in the absence of SMAD4 andregulates mammalian keratinocyte differentiation by binding to theregulatory sequences of the transcription factor genes Mad1 (Mxd1– Mouse Genome Informatics) and Ovol1, which induce epidermaldifferentiation. Transcriptional induction mediated by the complexof SMAD2, SMAD3 and IKK is independent of the kinase activityof IKK. Interestingly, invasive squamous cell carcinomas that areresistant to the tumor suppressor action of TGF have defectiveIKK that cannot enter the nucleus and act as a Smad co-factor(Marinari et al., 2008).

Genes that are either transcriptionally co-regulated at the samedevelopmental time, or in the same tissue, are referred to assynexpression groups (Niehrs and Pollet, 1999). One such group isthe inhibitor of differentiation (Id) family of genes, which like otherTGF-responsive synexpression groups, respond to TGF familymembers via specific regulatory sequences present in their genes(see Table S1 in the supplementary material) (Karaulanov et al.,2004). Synexpression groups also require specific Smad-interactingtranscriptional co-factors for their expression. For example, the


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mammalian FoxO transcription factors bind to Smads andcoordinate the regulation of 11 genes that define the cytostatic,apoptotic and adaptive signaling responses of keratinocytes to TGF(see Table S1 in the supplementary material) (Gomis et al., 2006a).The helix-loop-helix HHM (human homolog of Maid; CCNDBP1)protein regulates a specific synexpression group of cell cycle andcell migration regulators (see Table S1 in the supplementarymaterial), and, accordingly, regulates growth inhibition andmigration in mammalian epithelial cells in response to TGF, whilemediating other responses in different cells (Ikushima et al., 2008).

In the Drosophila wing imaginal disc, the BMP ligand Dppactivates Mad (an R-Smad) and Medea (its Co-Smad) (see Table 1),which then directly repress certain transcription factor genes,including the transcriptional repressor brinker (brk). This repressionof brk by Mad-Medea leads to the derepression of optomotor blind(omb; bifid – FlyBase), zerknüllt (zen) and spalt (sal), which encodetranscription factors that regulate the expression of othertranscription factors, to provide patterning and morphogeneticinformation to the developing wing (de Celis and Barrio, 2000; Shenet al., 2008). sal, however, additionally requires direct binding andtransactivation by the Mad-Medea complex.

In C. elegans, the sma-9 gene is involved in neuronalspecification within a restricted group of rays in the tail of thedeveloping worm and is expressed during early larval stages (Lianget al., 2003). SMA-9 is the ortholog of the Drosophila zinc-fingertranscription factor Schnurri, a co-factor of the Mad-Medea complexthat represses brk expression (Marty et al., 2000). By analogy withDrosophila, SMA-9 might mediate BMP-like DBL-1 signaling bycomplexing with SMA-2, SMA-3 or SMA-4. Indeed, SMA-9 actsas both a transcriptional repressor and an activator downstream ofDBL-1. Newly identified targets of this pathway are orthologs oftranscription factors that are already implicated in mammalian BMPsignaling, such as Runx and Fos, or orthologs of Hedgehog signalingproteins (Liang et al., 2007). Interestingly, mouse Schnurri-2(HIVEP2 – Mouse Genome Informatics) also regulatesTGF/BMP-dependent gene expression. It binds to SMAD1-SMAD4 and to the transcriptional co-factor C/EBP(CCAAT/enhancer-binding protein ) to induce the PPAR2(peroxisome proliferator-activated receptor 2; Pparg) gene thatregulates adipocyte differentiation (Jin et al., 2006). Thus, mice thatlack the Schnurri-2 gene have reduced fat.

Finally, as we discuss in Box 3, TGF family transcriptionalregulation in development also occurs via the regulation of micro-RNA (miRNA) genes, and via the reciprocal regulation of TGFsignaling by miRNAs.

ConclusionsAs TGF research continues with ever increasing speed, we foreseeimportant novel findings regarding the mechanisms of TGFreceptor regulation and specificity of signaling, cytoplasmictrafficking of receptors and Smads, nuclear dynamics of Smad-chromatin associations and their relationship to developmentalprocesses. The functional implications of ‘promiscuous’ signalingby TGF family receptor kinases that simultaneously activateTGF- and BMP-like Smad pathways, as well as MAPK and otherpathways, needs to be analyzed carefully and with quantitativemethods. The area of post-translational modifications of TGFreceptors and Smads will continue its prolific expansion. Moresensitive proteomic approaches will be useful in dissecting all thecomponents of signaling complexes and their dynamic nature,especially if coupled to multi-protein imaging in real time. Progressin the modeling of signaling dynamics and of the protein networks

that participate in the TGF family cascades should provide freshideas about new regulatory nodes in the network, and should alsodefine more quantitatively critical parameters that govern thebehavior of the network. A major challenge is to decipher the rolesof the TGF pathways during late stages of embryogenesis andduring neonatal life by conditional activation and inactivation ofTGF signaling components in model organisms. The importanceof cross-talk during different developmental stages between TGFand Wnt, Hedgehog, FGF or other pathways should be another focusfor future research. Finally, in the context of development, morecomplete circuits of target genes, and their corresponding protein orRNA regulators, will need to be delineated in the global effort toprovide a systems-level description of TGF pathways in everytissue and organ.

AcknowledgementsWe acknowledge funding by the Ludwig Institute for Cancer Research (LICR),the Atlantic Philanthropies/LICR Clinical Discovery Program, the SwedishCancer Society, the Swedish Research Council, the European Union MarieCurie Research Training Network ‘EpiPlastCarcinoma’ and the European UnionNetwork of Excellence ‘ENFIN’. We acknowledge the contribution of all pastand present members of the TGF signaling group to the scientific workemanating from our laboratory.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/22/3699/DC1

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